Ideal Extraction Temperature for Antioxidants from Holy Basil And

Progress in Nutrition 2019; Vol. 21, N. 4: 971-976 DOI: 10.23751/pn.v21i4.6179 © Mattioli 1885

Original articles

Ideal extraction temperature for antioxidants from holy basil and bunching onion Muhammad Muzaffar Ali Khan Khattak1,2,3, Adlina Zainal Abidin1, Nuraniza Azahari1 1Department of Nutrition Sciences, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia, Jalan Sultan Ahmad Shah, Bandar Indera Mahkota Kuantan, 25200, Pahang Darul Makmur, Malaysia - E-mail: [email protected]; 2Non Communicable Diseases Research Unit, Kulliyyah of Medicine, International Islamic University Malaysia (IIUM), Jalan Sultan Ah- mad Shah, Bandar Indera Mahkota, 25200 Kuantan, Pahang Darul Makmur; Malaysia; 3International Institute for Halal Research and Training (INHART), E5 2-2, Level 2, Block E5, Kulliyyah of Engineering, International Islamic University Malaysia, P.O. Box 10 Kuala Lumpur, Malaysia.

Summary. This study aimed to determine ideal temperature for antioxidants from holy basil and bunching onion. Holy Basil (Ocimum Tenuiflorum) and Onion (Allium Fistulosum) were extracted with various temperatures ranging from 75 – 100 °C with two solvents i.e. methanol and water (room temperature & boiling). Total phenolic contents (TPC) and total flavonoid contents (TFC) were determined in the extracts by using the Folin-Ciocalteau and Aluminum chloride complex formation assays respectively. Extracts were analyzed in triplicates statistically compared using one-way analysis of variance (ANOVA) and the difference between the mean was ascertained at 95% confidence interval (P<0.05) using Tukey›s honest significance test. In both holy basil and bunching onion, the TPC and TFC values for methanolic extracts were significantly (P<0.01, P<0.001) higher than the water extracts. The best temperature among the various temperature used was 85˚C where maximum TPC and TFC were observed in the extracts. This study shows that using optimum temperature helps in extraction of maximum antioxidants with methanol.

Keywords: culinary herbs, extraction temperature, antioxidants concentration

Introduction cals in human body (3) e.g. polyphenols (4). Phytos- terols, (5-7), flavonoids (8), vitamin C (1, 9-12). Apart The interest in the potential uses of natural anti- from health benefits, antioxidants also have many in- oxidants as food preservatives and for health reason has dustrial uses, such as preservatives in cosmetics and to shown an increasing trend (1). Over the past 20 years, prevent the degradation of rubber and gasoline (12). synthetic antioxidants such as butylated hydroxyani- However, synthetic antioxidants used in the food and sole (BHA), butylated hydroxytolulene (BHT) and pharmaceutical industries may be toxic (2) compared tert-butyl hydroquinone (TBHQ) are being used as to natural sources (13). Holy basil (Ocimum tenuiflorum food preservatives due to their low cost. However, or Ocimum sanctum) is an aromatic plant that is native several researchers have found that there are possible to Eastern World. It is also called tulsi in India and unhealthy effects of synthetic antioxidants and devel- is considered as a holy religious plant among Hindus. oped countries like Japan, Europe and Canada have Apart from this, it is used as therapeutic ingredients banned the use of synthetic antioxidants used in food for various purposes (14-16). Furthermore, commonly (2). There are numerous types of natural antioxidants used as a condiment and garnishing agent in variety which act as reducing agents to stabilize the free radi- of cuisines (17). According to (18), the antioxidants 972 M. M. A. K. Khattak, A. Z. Abidin, N. Azahari found in herbs have been identified to have multiple atures (65, 75, 85, 95 and 100˚C), water (room temper- biological effects, including antioxidant activity. The ature) and hot water (65, 75, 85, 95 and 100˚C). Two most important chemical constituents in O. tenui- assessments were performed namely Folin-Ciocalteau florum extract (mainly in leaf ) are eugenol, carvacrol, and Aluminium chloride complex formation for total tannins, methyl eugenol and caryophyllene (19-22). phenolic contents (TPC) and total flavonoid (TFC) However, other researchers have observed that other contents. For phenolic contents, a calibration curve essential antioxidants, such as ascorbic acid, carot- of the Gallic acid standards (5, 2.5, 1, 0.5, 0.1 mg/l) enoids, tocopherol, tocotrienols, glutathione, phenolic was prepared; the absorbance was measured at 760 compounds (like flavonoid) and cichoric acid are also nm using UV-Vis spectrophotometer and using pure present in it (1, 10, 23). Furthermore, O. tenuiflorum methanol as a blank as described previously by (38). also has showed to exhibit a hepatic protective effect The concentration of phenolic contents in samples was and can be used in the treatment of hepatic disorders estimated using the formula below: anti-stress, immune modulator, anti-inflammatory, mast cell stabilization, anti-histamine (14) and (24). Phenol content mg GAE/g = [(Slope x absorbance) + Onion (A. fistulosum) also possesses antioxidants c] / Sample concentration which are characterized by its higher contents of thio- *c is the y-intercept sulfunates (allicin), an antioxidant useful for disease condition (25, 26). Allicin also has anti-bacterial, anti- Total flavonoid contents were determined by Alu- viral, and anti-fungal properties (9, 25, 27, 28). Vari- minium chloride complex formation as described by ous studies report that cooking temperature may affect (39). A calibration curve of the quercetin standards (5, antioxidants concertation/activity (1, 3, 13, 29-33). 2.5, 1, 0.5, 0.1 mg/l) was prepared and the absorbance The optimum release depends on the types and nature was measured at 425 nm using UV-Vis spectropho- of the material and antioxidants (34). Various methods tometer and using pure methanol as a blank. The con- of extraction are used but little attention has been paid centration of total flavonoid in samples was estimated to have ideal temperature for extractions (35). Similar- using the formula below: ly, aqueous and organic solvents would also have posi- tive or negative effect on extraction (36). Therefore, it Flavonoid contents (QE mg/g) = [(Slope x absor- is would be excellent to have optimal solvent and ideal bance) + c] /Sample concentration temperature for extraction of antioxidants (37) *c is the y-intercept Despite of the wide uses of these two common herbs in cooking, the optimum temperature that would Statistical analysis optimize the retention of antioxidant contents of these Statistical analysis of the data was performed by herbs in cooking is still unknown. Thus, there is a need using SPSS (Version 12.1), statistically compared us- to study the effect of temperature to identify the suit- ing one-way analysis of variance (ANOVA) and the able temperature that can maximize the retention of difference between the mean was ascertained at 95% the antioxidants in the extract. confidence interval (P<0.05) using Tukey’s honest significance test.

Materials and Methods Results and Discussion This study involved two famous Asian culinary herbs, namely holy basil and bunching onion. Both The present study demonstrated some interesting herbs were purchased from the local market in Kuan- findings for the antioxidant concentration of extracts tan, Pahang, Malaysia. The herbs were then cleaned with different temperatures. As mentioned earlier, the with distilled water, dried and ground. After grinding, antioxidant concentrations of these two herbs were de- extracted separately with methanol at various temper- termined in the form of total phenolic contents (TPC) Ideal extraction temperature for antioxidants from holy basil and bunching onion 973 and total flavonoid contents (TFC) and extracted with lead to lower antioxidant concentration (40). The results two different solvent namely methanol and water. The of this study reveal that TPC at 85˚C was higher than at TPC of three different procedure extracts of the two 65, 75, 95 and 100 ˚C which might be the suitable tem- herbs i.e. are presented in the Figure 1 & 2 and Table 1. perature. Furthermore, increase in temperature beyond Highest (P < 0.001) concentration was obtained 85 ˚C would lead to a decrease in TPC yield as it has for the total phenolic contents at 85ºC for both herbs been observed at 95 and 100˚C (Fig 1). Similar, effect compared to the rest of the temperatures used. From has been suggested by (13) that an increase in tempera- these results, it can be deduced that hot water extracts at ture will lead to an increase in TPC to the maximum 85˚C exhibits higher content of TPC (Fig. 1). General concentration whereas there will be a decrease at further concept of the effect of the temperature on the antioxi- increase of temperature and that is what we observed dant’s concentration is that higher temperature would (85 ˚C). The present study reveals that the effect of tem-

Figure 1. Alteration in cooking temperature affects total phenol Figure 2. Alteration in cooking temperature affects total flavo- contents of holy basil and onion. Each bar represents the mean noid contents of holy basil and onion. Each bar represents the values ± standard deviation, where n = 3. mean values ± standard deviation, where n = 3.

Table 1. The phenolic contents of these extracts were estimated using a standard curve of Gallic acid and expressed as milligrams of Gallic acid equivalents (GAE), the flavonoid contents that were projected using a standard curve of quercetin and expressed as mg of Quercetin equivalents (QE) over weight of sample in gram and Holy Basil Extracts Onion Extracts Methanol Water Hot Water Methanol Water Hot Water (room Temperature) (room Temperature) Total Phenol Contents (TPC) (mgs GAE/g) 0.83 ± 0.17a 0.02 ± 0.04b 0.11 ± 0.01b 4.16 ± 0.63c 0.01 ± 0.03b 0.11 ± 0.03b Total Flavonoid Contents (TFC) (mg of Quercetin equivalents (QE) 5.04 ± 0.06a 1.57 ± 0.01c 3.77 ± 0.05b 4.91 ± 0.12b 2.14 ± 0.04c 4.81 ± 0.11a Each value represents the mean ± standard deviation, where n = 3. The vales with different superscripts are significantly (P<0.05, P<0.01) different. 974 M. M. A. K. Khattak, A. Z. Abidin, N. Azahari perature on the antioxidant contents of Holy basil and (46), who found that the phenolic compounds yield Onion were different from one temperature to another 30% more with methanol compared to the other forms which is due to the thermal effects which varies depend- of solvents. The content of holy basil and onion show ing on the type of plants, species (33, 41, 42). that both herbs are important for antioxidant activities The TFC was higher at 95 and 100˚C of hot wa- (10). However, the concentration of the TFC is varied ter extracts as compared to other temperatures for depending on the type of extraction solvent. Similar to both herbs (Figure 2). Similar result was found by the TPC, the highest concentration of TFC was ob- (10) who reports that heating at a higher temperature served in the methanol extracts of Holy basil and On- gave a higher flavonoid content compared to heating ion. A similar observation has been reported for spices at a lower temperature. This has been attributed to the with methanol to be the most effective extraction agent hydroxyl structure of flavonoids that is effectively ex- for TFC compared to the other solvents (10). tracted at 95˚C (33). Surprisingly, the results of this Phenolic compounds are good sources of antioxi- study showed that the highest content of TFC was ob- dants and have excellent potential in elucidating an- tained at higher temperature compared to TPC. This tioxidants scavenging activity (18, 44, 45). However, was similar to (13) who found that the highest yield of the concentration of the phenolic compounds varies TFC higher temperature (63ºC) while the highest yield depending on the type of extraction (46, 47) and (10). of TPC were observed at lower temperature (53ºC). The observed concentration TPC and TFC may have These authors attribute the results to the differences in the antioxidant activity and free radical-scavenging the hydroxyl group in the phenolic compounds which capacity in the tested herbs (44, 48, 49) and (44). In responsible for responding differently towards thermal fact, the higher the phenolic contents, the higher the treatment. The specific hydroxyl structure in flavonoids free radical-scavenging activity and antioxidant ca- and are acting in a specific different mechanism (43). pacity of the sample, and vice versa (49, 50). In the The variation may be due to the differences in cultivar present study, it was found that extraction solvent also which may be influenced by genetic factors (41) and influenced the antioxidant contents of these two herbs. varied from one region to another (44). Methanol possessed highest yields of total phenols From Figure 1 Holy basil expressed the highest as compared to hot and cold water. Interestingly, the phenolic contents at temperature of 85˚C. As suggest- present study revealed that the effect of temperature ed by (44), the total phenolic content which mainly on the antioxidant contents of both herbs were unique affects the antioxidant activity is a main parameter to from one temperature to another. Hot water extracts at determine the overall concentration and activity of 85˚C had the highest phenolic compounds followed by antioxidants in the herbs. Therefore, it is considerably 65, 95, 100 and 75˚C for Holy basil while for Onion; valid to conclude that the temperature which resulted the phenolic compounds yield ranged from highest to in the highest phenolic compounds was the optimum lowest was at 85, 65, 75, 95 and 100˚C. In fact, the an- temperature for retention of the antioxidant contents tioxidant activity elucidated by Holy basil of hot water in the tested samples. Figure 1 has revealed that 85˚C extracts at all manipulated temperatures was consider- was the optimum temperature for the optimization of ably higher except for 95˚C. Thus, it appears that 85˚C antioxidants concentration in Holy basil. was the optimum temperature for the optimization of These results show that both tested herbs pos- antioxidants contents for both herbs. sess significant amount of phenolic compounds. The content of TPC was highest in methanol compared to cold and hot water extract similar to the other studies Acknowledgements of Holy basil (45) and Onion (44). It can be observed from these results that methanol extracts of both herbs The Authors thankfully acknowledge the support of Kulliyyah yielded the highest concentration of TFC compared to of Allied Health Sciences, International Islamic University, Malaysia, Jalan Istana, Bandar Indera Mahkota 25200 Kuantan, hot and cold water extracts respectively. This has been Pahang Darul Makmur, Malaysia reported elsewhere in a study conducted by a group of Ideal extraction temperature for antioxidants from holy basil and bunching onion 975

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